Volume 12, Issue 4, Pages 558-570 (October 2012) Short-Range Exosomal Transfer of Viral RNA from Infected Cells to Plasmacytoid Dendritic Cells Triggers Innate Immunity  Marlène Dreux, Urtzi Garaigorta, Bryan Boyd, Elodie Décembre, Josan Chung, Christina Whitten-Bauer, Stefan Wieland, Francis V. Chisari  Cell Host & Microbe  Volume 12, Issue 4, Pages 558-570 (October 2012) DOI: 10.1016/j.chom.2012.08.010 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Inhibition of pDC Activation Triggered by HCV-Infected and SGR Cells by Exosome Release Inhibitors (A–C) Quantification of IFN-α in supernatants of pDCs cocultured with HCV-infected Huh-7.5.1c2 cells (moi of 1 for 48 hr) that were treated or not with exosome-release inhibitors GW4869 or spiroepoxide (10 and 5 μM, respectively) throughout the course of the coculture (A). Uninfected Huh-7.5.1c2 cells are referred to as control (cont) cells. The hatch marks (#) indicate results below the limit of detection of the IFN-α ELISA (i.e., 12.5 pg/ml). In parallel, the intracellular HCV-RNA levels (B) and extracellular infectious virus production (C) of HCV-infected cells treated with exosome release inhibitors were analyzed. Results are representative of two independent experiments, each performed in triplicate. Error bars represent the mean ± SD. (D) Quantification of IFN-α in supernatants of pDCs cocultured with HCV-SGR Huh-7.5.1c2 cells after treatment with exosome-release inhibitors GW4869 or spiroepoxide (treatment with 10 and 5 μM, respectively) throughout the course of the coculture. HCV-negative Huh-7.5.1c2 cells served as controls (cont cells). (E) Intracellular HCV-RNA levels in HCV-SGR cells treated for 24 hr with exosome release inhibitors. (F) Parallel pDC/control cell cocultures were incubated with the TLR-7 ligand agonist R848 and supernatants were analyzed for IFN-α. Results in (D)–(F) are representative of three independent experiments, each performed in triplicate. Bar graphs depict the mean ± SD. The treatment conditions (i.e., incubation time and concentration) were exactly the same for the analysis of IFN-α production, HCV-RNA replication, and extracellular infectious virus production. See also Figure S1. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 HCV RNA Is Transferred from HCV-SGR Cells to pDCs (A) Specificity of HCV-RNA detection (green) by FISH in HCV-infected Huh-7.5.1c2 cells that expressed high levels of HCV RNA (left panel), that were treated with the HCV polymerase inhibitor 2′-C-methyladenosine (5 μM for 24 hr; middle panel) and in uninfected Huh-7.5.1c2 cells (no HCV; right panel). Nuclei are stained with Hoechst dye (blue). (B and C) Projection (B) and consecutive Z-axis sections (C) of pDC cocultured with HCV-SGR Huh-7.5.1c2 cells (3.5-fold magnification of the white box in [B]). Green: HCV RNA; red: IFN-α protein; blue: nuclei. HCV RNAs inside pDC are indicated by white arrows. Of note, FISH HCV-RNA signals are lower in HCV-SGR cells because they contain ∼10-fold less HCV RNA than infected cells, and in addition, FISH signals are reduced when combined with immunofluorescent staining (data not shown). Similar results were obtained in five independent experiments. (D) Summary table showing the fraction of total cocultured pDCs that contain HCV RNA. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Colocalization of HCV RNA, Exosome Markers in HCV-SGR Cells (A–C) Detection of HCV RNA (red); CD63 (A, green), eGFP-CD81 (B, green), and FLAG-CHMP4B proteins (C, green); and nuclei (blue) in HCV-SGR Huh-7.5.1c2 cells. Panels 2–4 correspond to a 3.6-fold magnification of the white box in #1 in each row. The arrows indicate HCV RNA in CD63-, CD81-, or CHMP4B-positive compartments. The frequencies of HCV-SGR cells in which HCV RNA colocalized with CHMP4B, CD81, and CD63 were 25/29, 38/56, and 17/24, respectively. Results are representative of three independent experiments. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 RNase-Resistant HCV RNA Is Secreted from HCV-SGR Cells (A–C) HCV-RNA levels in precleared supernatants from HCV-SGR Huh-7.5.1c2 cells (A: n = 4, mean ± SD; C: n = 2, mean ± SD), and in vitro transcribed HCV RNA (B: n = 3, mean ± SD) were treated with RNase, NP40, and/or RNase inhibitor, as indicated. The hatch marks (#) indicate results below the detection limit of the assay (i.e., 500 HCV GE/ml). GE, genome equivalents. (D) HCV and GADPH RNA levels in precleared supernatants from HCV-SGR Huh-7.5.1c2 cells (extracell) either untreated or treated with RNase, NP40, and untreated SGR cell RNA (intracell) as indicated. Error bars represent the mean ± SD (n = 3); paired Student’s t test, ∗∗p < 0.005. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Exosomes Prepared from HCV-SGR Cell Supernatants Trigger IFN-α Production by pDCs (A) Comparison of exosome (CD63 and CD81) and nonexosome (EEA-1, calnexin, and COX IV) markers in exosomes prepared from HCV-SGR Huh-7.5.1c2 cell supernatants and from the corresponding whole-cell lysates (intracell) by immunoblotting. The molecular weight markers (MW, in kDa) are indicated to the right. (B) Exosome preparations were treated with NP40 and/or RNase and qRT-PCR results are expressed as HCV GE or GAPDH mRNA copies per μg of total protein. Error bars represent the mean ± SD (n = 3). (C) Northern blot analysis. The input copy number of HCV and GADPH RNA determined by qRT-PCR is indicated below each blot. Huh-7.5.1c2 cells served as negative control (cont cells). NA, not applicable. (D) pDCs (4 × 105 cells) were incubated with concentrated exosomes at varying HCV GE/pDC ratios or with R848, a TLR7 agonist. The hatch marks (#) indicate results below the limit of detection of the IFN-α ELISA (12.5 pg/ml). Results are representative of two independent experiments. Error bars represent the mean ± SD. See also Figure S2. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 ESCRT Proteins Are Required for Production of the pDC-Activating Signal by HCV-SGR and HCV-Infected Cells (A and B) The impact of shRNA-mediated CHMP4B downregulation, using two different shRNAs (#30 and #69, described in Table S1) (A) and shRNA-mediated TSG101 downregulation (B) on expression of the corresponding proteins (upper panels), levels of IFN-α in HCV-SGR Huh-7.5.1c2 cell-pDC coculture supernatants (middle panels), and intracellular HCV RNA in Huh-7.5.1c2 SGR cells (lower panels) was compared with HCV-SGR cells transduced with shRNA against GFP and an shRNA against an unrelated cellular protein (Atg4B) or nontransduced HCV-SGR cells (−). HCV-negative Huh-7.5.1c2 cells served as controls (cont cells). The hatch marks (#) indicate results below the limit of detection of the IFN-α ELISA (12.5 pg/ml). Results are representative of two independent experiments, each performed in triplicate. Error bars represent the mean ± SD. NS, nonspecific band; PR, Ponceau red staining; MW, molecular weight marker. (C–E) CHMP4B and TSG101 downregulated Huh7.5.1c2 cells, infected by HCV (moi of 5 for 48 hr) were cocultured with pDCs for 20 hr. The levels of CHMP4B and TSG101 expression (C), IFN-α in coculture supernatants (D, upper panel), intracellular HCV RNA (D, lower panel), and titration of HCV infectious particles in supernatants of the infected cells (E) were compared with control HCV-infected cells transduced with shRNA against GFP. Uninfected Huh-7.5.1c2 cells served as controls (cont cells). Results are representative of two independent experiments, each performed in triplicate. Error bars represent the mean ± SD. See also Figure S3. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 ANXA2 Is Required for HCV-Infected and SGR Cells to Trigger IFN-α Production by pDCs (A–C) shRNA-mediated downregulation of ANXA2 protein in HCV-SGR Huh-7.5.1c2 cells (A) strongly reduced IFN-α production by cocultured pDCs (B), but had no impact on HCV replication (C). All results were compared with ANXA2 shRNA-transduced HCV-SGR Huh-7.5.1c2 cells that ectopically express an shRNA-resistant variant of ANXA2 (Rescue). Results are representative of two independent experiments, each performed in triplicate. NS, nonspecific band; MW, molecular weight markers. (D–G) Levels of downregulation of ANXA2 expression in HCV-infected Huh-7.5.1c2 cells (moi of 5 for 48 hr) (D), IFN-α in HCV-infected cell-pDC coculture supernatants (E), intracellular HCV RNA in HCV-infected cells (F), and HCV infectious particles in supernatants of the infected cells (G) were compared with HCV-infected cells transduced with an shRNA against GFP. HCV-negative Huh-7.5.1c2 cells served as controls (cont cells). The hatch marks (#) indicate results below the limit of detection of the IFN-α ELISA (12.5 pg/ml). Error bars represent the mean ± SD. See also Figure S4. Cell Host & Microbe 2012 12, 558-570DOI: (10.1016/j.chom.2012.08.010) Copyright © 2012 Elsevier Inc. Terms and Conditions